During early development in vertebrates, including humans, determination and differentiation of axial skeletal muscles and vertebral elements is controlled by complex processes of embryonic pattern formation. As part of these pattern forming processes, primordial cells flanking the neural tube and notochord, called the presomitic mesoderm, mature into early segmental structures called the somites. During somite maturation the presomitic mesoderm buds into segments to form the epithelial somites, this process proceeds in a cranio-caudal direction according to an intrinsic developmental timetable (reviewed by Keynes and Stern, Development 103: 413-429, 1989; Tam and Traynor, Anat. Embryol. 189: 275-305, 1994). After the somites are formed, the ventral-medial parts of the somites delaminate to form the sclerotome, while the dorsal-lateral component of the somite forms the dermomyotome. Prior to this stage of development, the fate of cells in the epithelial somites is plastic, or "undetermined," whereas after this stage the sclerotome cells are "committed" to differentiate into the vertebral column and ribs, and dermomyotome cells are committed to form dermis and axial skeletal muscle.
Accumulating evidence suggests that the patterning of somites into sclerotomal and dermomyotomal compartments depends on inductive signals from other cells, particularly cells forming the notochord and floor plate. A key inductive signal in this regard may be provided by the gene Sonic hedgehog, which has been shown to enhance sclerotomal marker expression and repress dermomyotomal marker expression when the gene is expressed ectopically or in heterologous cells (Johnson et al., Cell 79: 1165-1173, 1994; Fan et al., Cell 79: 1175-1186, 1994). Another important set of determinants is the MyoD family of myogenic factors which appear to act "downstream" (i.e., subordinately in a developmental regulatory hierarchy) of Sonic hedgehog in determining somite cell fate. The MyoD family of genes includes myoD, myf5, myogenin and MRF4, which each encode muscle specific transcriptional regulatory factors belonging to the basic-helix-loop-helix (bHLH) class of DNA binding proteins (see reviews by Emerson, Curr. Biol. 2: 1065-1075, 1990; Weintraub, et al., Science 251: 761-766, 1991).
All of the MyoD family of myogenic factors share the remarkable property of being able to convert cells into a myogenic differentiation pathway when the cells are transfected with a MyoD family member gene. For example, primary fibroblasts of different species transfected with the myoD gene are induced to express muscle specific genes, and in many cases form muscle fibers and differentiate into myoblasts or myotubes (see for example, Weintraub et al., Proc. Natl. Acad. Sci. USA 86: 5434-5438, 1989). The myogenic activity of the MyoD family genes is explained in part by their conservative, bHLH domains, which includes a basic region required for DNA binding, and an HLH region required for dimerization (see for example, Davis et al., Cell 60: 733-746, 1990). Further explanation for the myogenic activity of MyoD family genes includes their ability to heterodimerize with "E proteins" and the ability of these heterodimeric complexes to bind to the "E box" sequence motif, CANNTG, of many muscle specific genes and transactivate their expression (reviewed by Kadesh, Imm. Today 13: 31-36, 1992).
The fact that cell commitment toward skeletal muscle differentiation is determined cell-autonomously by myoD, myf5, myogenin and MRF4 raises important questions concerning the "upstream" control of MyoD genes and/or their encoded transcription factors. The ability of MyoD family genes to overcome preexisting cell fates when expressed ectopically indicates that precise regulatory control of the MyoD genes is essential for normal development. This regulation likely includes mechanisms that inhibit expression and/or function of MyoD family genes and/or their products in the sclerotome, because cells in this embryonic compartment are derived from the same precursors as the myotome but do not undergo myogenesis. Such negative regulatory mechanisms controlling MyoD family gene expression, and/or MyoD myogenic factor activity have heretofore remained largely unexplored, and many fundamental questions remain concerning this aspect of myogenic regulation.
Accordingly, there is a general need in the art for further discovery and characterization of myogenic regulatory factors affecting normal and abnormal development in vertebrates. In particular, a general need exists for discovery and characterization of factors involved in regulating the expression and/or activity of MyoD family genes and/or the myogenic factors they encode. In addition to these fundamental needs, there also remain more specific needs in the art to develop effective tools to model, diagnose and treat defects in myogenesis responsible for abnormal development and disease conditions in mammals, including humans.